AP Biology : 4.7 Regulation of Cell Cycle- Study Notes

D. Biotechnology
➢ Recombinant DNA generated by combining DNA from multiple sources to create a unique DNA molecule that is not found in nature
○ Ex. introduction of a eukaryotic gene of interest into a bacterium for production
➢ Polymerase Chain Reaction (PCR)

  • Enables the creation of billions of copies of genes within a few hours
  •  DNA polymerase, DNA, and lots of nucleotides added in a small PCR tube
  •  Thermocycler
    ■ PCR machine that heats, cools, and warms PCR tubes many times
    ■ Each time the machine is heated, the hydrogen bonds break, separating the double-stranded DNA (Denaturation)
    ■ As it cools, primers bind to the sequence flanking the region of the DNA we want to copy, primers can form hydrogen bonds with ends of target sequence (Annealing)
    ■ When it is warmed, polymerase binds to the primers on each strand and adds
    nucleotides on each template strands (extension)
    ■ REPEAT exponentially

➢ Transformation

  •  Transformation: process of giving bacteria foreign DNA
    ■ Genes of interest (vectors) placed into small circular DNA molecule called a plasmid
               ● Plasmid usually codes for antibiotic resistance
               ● Small ring of DNA found in bacteria that is replicated separately form the chromosomal DNA
               ● Not in all bacteria
    ■ 1. Extract the plasmid
    ■ 2. Add a restriction enzyme that will cut the ring open
               ● Restriction enzymes usually used to cut up foreign DNA, but are used by
    scientists for this purpose
               ● Cuts palindromes, leaving “sticky ends”
               ● Always cut at the same nucleotide sequence
    ■ 3. Cut a piece of human DNA with same restriction enzyme
              ● Reverse transcriptase must be used to process DNA since prokaryotes do not have RNA processing to remove introns
  •  DNA transcribed and mRNA is processed, and then reverse transcriptase turns mRNA back into DNA
            ● Reverse transcriptase also used by retroviruses
            ● Problems: vector may be too big, and there is no direct way to force the plasmid to accept the vector
    ■ 4. Mix the cut plasmids with the cut human DNA-some will align right due to their sticky ends
          ● Ligase used to glue ends back together
    ■ 5. Allow bacteria to take plasmid back in
         ● Heat shock/electric shock used to change membrane so plasmid can reenter easily
    ■ 6. Allow to reproduce

■ Not all bacteria will be transformed, can be tested by using antibiotic resistance
■ Allows the safe mass-production of proteins used for medicine
■ Important role in the study of gene expression
Transfection: putting a plasmid into a eukaryotic cell, rather than a bacteria cell
➢ Gel Electrophoresis

  •  DNA fragments can be separated according to their molecular weight using gel electrophoresis
  •  DNA put into wells on negative end, and when a current is run through the gel, the DNA moves across gel according to their weight
  •  Because DNA and RNA are negatively charged, they migrate through the gel toward the positive pole of the electrical field
    ■ Smaller fragments move faster and farther
  •  Restriction enzymes used to create a molecular fingerprint
    ■ Places where enzymes cut and thus the sizes are unique for each person

➢ Stem cells also very important in biotechnology since they can turn into many different kinds of cells, but it is controversial due to harvesting methods

  • Totipotent cell: capable of giving rise to any type of cell or a complete embryo
  • Pluripotent cell:capable of giving rise to different cell types


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